PROJECT SUMMARY The Candidate is a postdoctoral fellow committed to developing an academic research group focused on applying bioinformatics to understand the pathophysiology of alcohol-related liver diseases (ALD). His previous and current postdoctoral work has given him the unique skillset to answer questions involving gene expression/regulation in the immune system. The Career Development Plan describes 2 years of mentored research wherein the candidate will develop skills in clinical immunology, generate the sequencing data outlined in the proposal, and learn leadership skills to transition into independence. The next 3 years, after obtaining an independent faculty position, will be dedicated to developing new data analysis pipelines and establishing cell biological and bioinformatics tools to understand gene regulation in the innate immune system, which will allow the candidate to establish future projects in ALD immunology. Research Plan: ALD is a spectrum of disorders that affect a growing number of people worldwide. Alcoholic Hepatitis (AH) is a severe inflammatory disease that can increase the morbidity and severity of ALD disorders. In AH, the innate immune system is hypersensitive to microbial byproducts. Alcohol consumption causes gut-barrier disruption, leading to leakage of gut microbes into the portal circulation. The liver immune system is able to detect these microbes through pattern recognition receptors, including the Toll-like receptors (TLRs) and C-type Lectin receptors (CTLs). While TLRs have been well studied for their role in sensitizing the innate immune cells to microbial products, the CTLs have only recently been implicated in mouse models of ALD. CTLs are a large family of PRRs that sense a vast diversity of microbes, including bacteria, fungi, and viruses. Our work has found that many members of the CTL gene family are upregulated in the liver and peripheral blood mononuclear cells PBMCs of AH patients. CTLs were also upregulated in PBMCs in response to LPS/TLR4 signaling. We predict CTLs are upregulated in response to gut- derived LPS in order to increase sensing for other microbes that may be present in the blood, making this pathway a secondary innate immune surveillance pathway. In this proposal, we will test this hypothesis in three aims to address the functional role and regulation of this immune surveillance pathway, and the mechanism by which it is exacerbated in AH. First, we will use PBMCs isolated from AH patients to measure increased sensitivity to CTL agonists, as well as micro-/mycobiome sequencing to determine what microbial byproducts were in circulation in patients. Second, we will use single-cell RNA sequencing (scRNA-seq) to dissect the different monocyte subclasses, variation in CTL expression, and how different cell types respond to CTL agonists. Third, because CTL genes are clustered in the genome, we will use scRNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin) to understand co-regulation of nearby genes in the genome. Altogether, this proposal will further our understanding of CTL mediated immune surveillance in host/microbial interactions during AH disease progression and potentially identify new therapeutic targets to decrease inflammation.